Abstract: Apoptosis is a form of programmed cell death that plays a crucial role in normal tissue homeostasis and development. In response to various genotoxic or cytotoxic stressors, apoptosis occurs by release of cytochrome c from the mitochondria, which promotes nucleation of a structure known as the apoptosome to activate cysteine proteases called caspases. In contrast to untransformed cells, cancer cells exhibit marked resistance to apoptosis by blocking mitochondrial cytochrome c release and/or by inhibiting apoptosome formation following cytochrome c release. Overexpression of the receptor tyrosine kinase ErbB2 is known to be associated with poor prognosis in breast cancer patients. Despite the availability of ErbB2-specific molecular inhibitors, the majority of ErbB2- overexpressing breast cancers eventually develop resistance to these inhibitors over a period of time (i.e., acquired resistance), suggesting the presence of currently uncharacterized mechanisms of chemoresistance. In this proposal, I investigate the molecular mechanisms contributing to apoptotic inhibition in breast cancers with acquired resistance to lapatinib, an FDA-approved ErbB2 inhibitor. It is my central hypothesis that inhibition of apoptosome formation plays a critical role in acquired lapatinib resistance in breast cancer. This hypothesis was formulated based on evidence from our laboratory as well as from others demonstrating that apoptosome inhibition can occur by two key independent pathways: HSP90 hypophosphorylation and CAS downregulation. Apoptosome inhibition by these mechanisms may override cell death signals caused by various pro-apoptotic stimuli. Towards this end, I will pursue the following two specific aims: 1) to determine the role of HSP90 hypophosphorylation in acquired lapatinib resistance, and 2) to elucidate the molecular mechanism of CAS downregulation. My primary career goal is to obtain a tenure-track position in an academic setting at a major U.S. university. My long-term goal is to build an independent research career as a molecular cancer biologist. To achieve these goals, I hope to both develop my intellectual knowledge base as well as increase my technical skill repertoire throughout the duration of the proposed study. This training should be readily attainable in the Kornbluth laboratory at Duke University Medical Center; our lab has considerable expertise in the biochemical analysis and molecular dissection of complex signaling pathways, both during apoptosis and cell cycle progression. In addition, I have obtained two outstanding collaborators, Drs. Neil Spector and Mark Dewhirst, who are established investigators in the fields of breast cancer and mouse xenograft models of cancer, respectively. Moreover, in order to promote and assure my progress during the Career Development Award period, I have organized an advisory committee consisting of well-established senior scientists with expertise in different areas relevant to my research and career goals. Collectively, the proposed studies will provide significant insight into mechanisms of chemoresistance and potential strategies for sensitizing cells to pro-apoptotic agents. Moreover, this work will provide me with the means to establish myself as an independent investigator.